Techniques are currently available for propagating foreign DNA segments in mammalian cells using SV40 and other papovaviruses as cloning vectors. The foreign DNA may be inserted into the late region of SV40; 1. between two appropriate restriction endonuclease sites; 2. by the poly(dA:dT) joining procedure; or 3. by joining at one endonuclease site and allowing for circularization in vivo. If the size of the inserted DNA segment is less than or equal to the fragment excised from the late gene region, one may co-infect permissive cells with a tsA helper viral DNA and hybrid and helper genomes will be packaged into virions at the non-permissive temperature. An analogous approach to this involves insertion of the foreign DNA into the early gene region of SV40 DNA and the subsequent cloning with a late gene ts mutant helper (tsB or C). Alternatively, one may construct hybrids larger than wild-type SV40 and propagate these molecules as free-replicating episomes in appropriate permissive or semi-permissive host cells. A limitation with this approach at present is the difficulty in the selection of cloned cell lines which retain viability, express the SV40 T-antigen and continue to replicate non-rearranged recombinant viral genomes. We have commenced work aimed at development of improved eukaryotic cloning vectors. Our approach is to insert the thymidine kinase gene of Herpes Simplex virus type I into the late gene region of polyoma virus or SV40 and then use tk-host cells for selection of these vectors. We have attempted to propagate an SV40-argF hybrid which contains approximately 1100 base pairs more than wild-type SV40 DNA by insertion of the 1650 base pair argF segment from E. coli between the EcoRI and Bam HI sites on the SV40 genomes. A significant fraction of transfected cells (5%) became T-antigen positive and were presumably replicating the argF-SV40 hybrid. However, this episome was lost from these cells after a few weeks.